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. Author manuscript; available in PMC: 2013 Sep 25.
Published in final edited form as: Cell. 2011 Nov 11;147(4):815–826. doi: 10.1016/j.cell.2011.09.050

Adipocyte NCoR Knockout Decreases PPARγ Phosphorylation and Enhances PPARγ Activity and Insulin Sensitivity

Pingping Li 1, WuQiang Fan 1, Jianfeng Xu 1, Min Lu 1, Hiroyasu Yamamoto 2, Johan Auwerx 2, Dorothy D Sears 1, Saswata Talukdar 1, DaYoung Oh 1, Ai Chen 1, Gautam Bandyopadhyay 1, Miriam Scadeng 3, Jachelle M Ofrecio 1, Sarah Nalbandian 1, Jerrold M Olefsky 1,*
PMCID: PMC3783197  NIHMSID: NIHMS333422  PMID: 22078880

Summary

Insulin resistance, tissue inflammation and adipose tissue dysfunction are features of obesity/Type 2 diabetes. Accordingly, we generated adipocyte-specific Nuclear Receptor Corepressor (NCoR) knock-out (AKO) mice to investigate the function of NCoR in adipocyte biology and glucose/insulin homeostasis. Despite increased obesity, glucose tolerance was improved in AKO mice, and euglycemic clamp studies demonstrated enhanced insulin sensitivity in liver, muscle and fat. Adipose tissue macrophage infiltration and inflammation were also decreased. PPARγ response genes were upregulated in adipose tissue from AKO mice and CDK5-mediated PPARγ ser-273 phosphorylation was reduced, creating a constitutively active PPARγ state. This identifies a novel function of NCoR as an adaptor protein which enhances the ability of CDK5 to associate with and phosphorylate PPARγ. The dominant function of adipocyte NCoR is to transrepress PPARγ and promote PPARγ ser-273 phosphorylation, such that NCoR deletion leads to adipogenesis, reduced inflammation, and enhanced systemic insulin sensitivity, phenocopying the TZD treated state.

Keywords: nuclear corepressor, insulin resistance, obesity, macrophage, adipogenesis

Introduction

The adipocyte uses well regulated transcriptional programs to adapt to environmental inputs through storage of calories as triglycerides and secretion of adipokines and other factors (Rosen and Spiegelman, 2006). PPARγ is a key factor controlling the importance of adipose tissue in whole-body glucose metabolism (Evans et al., 2004; Lehrke and Lazar, 2005; Saltiel and Olefsky, 1996; Sugii et al., 2009; Tontonoz and Spiegelman, 2008). PPARγ is a member of the nuclear hormone receptor (NR) family and is highly enriched in adipose tissue, where it plays a critical role in adipocyte differentiation, insulin sensitivity and adipokine/cytokine secretion (Evans et al., 2004; Imai et al., 2004; Rangwala and Lazar, 2004; Tontonoz and Spiegelman, 2008). Although its endogenous ligand is poorly understood, PPARγ is the molecular target for the thiazolidinedione (TZD) class of insulin sensitizing drugs used to treat type 2 diabetes.

Transcriptional control by NRs, including PPARγ and others, depends on multiprotein coregulatory complexes (Feige and Auwerx, 2007; Fowler and Alarid, 2004; Hermanson et al., 2002). In general, corepressor complexes are recruited to NRs in the absence of ligand, whereas, coactivator complexes are recruited to NRs in the presence of agonists (Lonard and O'Malley, 2005). Coactivators and corepressors modulate gene transcription by a variety of mechanisms, including histone acetylation, chromatin remodeling, and direct interactions with basal transcription complexes (Collingwood et al., 1999). There are several co-activators, such as CBP, PGClα, and CRTC2, which are known to play important roles in metabolic control (Handschin and Spiegelman, 2008; Revilla and Granja, 2009; Wang et al., 2010). However, the role and underlying mechanisms of corepressor function in metabolic tissues remains unclear. Two major NR corepressors are the silencing mediator of retinoid and thyroid hormone receptors (SMRT) and the nuclear receptor corepressor (NCoR) (Chen and Evans, 1995; Horlein et al., 1995). It has been shown that down-regulation of SMRT and NCoR expression in 3T3-L1 cells leads to enhanced adipocyte differentiation, in part through increased PPARγ transcriptional activity (Yu et al., 2005). However, their role in adipogenesis, adipocyte function, and glucose metabolism in vivo remains uncertain. Since whole body NCoR deletion is embronically lethal (Jepsen et al., 2000), we generated adipocyte specific NCoR knockout (AKO) mice to assess the role of this corepressor in glucose metabolism, insulin sensitivity, and adipogenesis. We show that AKO mice develop increased adiposity on HFD relative to WT controls. Despite this increase in obesity, the AKO animals exhibit enhanced systemic insulin sensitivity, improved glucose tolerance, and decreased adipose tissue inflammation. Taken together, these features phenocopy the effects of systemic TZD treatment.

Results

NCoR deletion in adipocytes

To investigate the specific role of adipocyte NCoR on adipogenesis and on the development of insulin resistance in response to HFD feeding, we generated adipocyte specific KO mice (AKO) using the Cre-lox system (NCoRfl/fl; aP2-Cre+/−) (Figures 1A and B). As controls, floxed NCoR mice that do not express Cre recombinase (Cre) were used (NCoRfl/fl; ap2-Cre−/−), referred to, hereafter, as WT. As expected, NCoR expression was greatly diminished in the epididymal adipose tissue of AKO mice (Figure 1C). However, since aP2/Fabp4 can also be expressed in macrophages, we determined whether aP2 Cre-mediated deletion of NCoR could be detected in this cell type. There was no decrease in NCoR expression in intraperitoneal (IP)-macrophages from the AKO mice (Figure 1C), consistent with previous studies using this ap2-cre mouse line, showing adipocyte restricted expression(He et al., 2003; Qi et al., 2009; Sabio et al., 2008; Sugii et al., 2009). This explains the ∼70% decrease in NCoR expression in whole epididymal adipose tissue, since NCoR is not deleted in macrophages or other non-adipocyte cell types present in this tissue. Interestingly, the magnitude of NCoR depletion in subcutaneous WAT and BAT was closer to 90%, most likely reflecting a lower amount of non-adipocytic cells, such as immune cells, in these depots. As also shown in Figure 1D, there was no NCoR deletion in any other tissue examined. SMRT is another corepressor, which in some contexts can function similarly to NCoR, but there were no changes in SMRT expression in the AKO mice in any tissues (Figure 1E).

Figure 1. NCoR targeting strategy and adipocyte-specific deletion.

Figure 1

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(A) Shown (top to bottom) are wild-type, floxed, and deleted NCoR gene loci. Primers used to distinguish WT and floxed alleles and sizes of the expected PCR products are indicated. (B) Genotyping results of wild type +/+, f/+, and f/f mice. (C) Relative messenger RNA levels of NCoR in adipose tissue and macrophages. Relative NCoR (D) and SMRT (E) mRNA levels in various tissues. Values are fold induction of gene expression normalized to the housekeeping gene Gapdh and expressed as mean ± SEM, n=8-10 in C- E, * P<0.05, ** P<0.01 for AKO versus WT. See also Table S2.

AKO mice are more obese than WT after HFD feeding

To investigate the functional significance of adipocyte specific NCoR deletion, both WT and AKO mice were fed a 60% high fat diet (HFD) for up to 17 weeks, starting at 8 weeks of age. As expected, WT mice became obese, but the AKO mice were even more obese as shown in Figure 2A. Thus, the body weight of the AKO mice was ∼15% greater than WT (Figure 2B) and this was accompanied by a 10% increase in food intake (Figure 2C). To further assess body composition changes accompanying this increase in obesity, MRI analyses were performed. As shown in Figures 2D-F, the AKO mice exhibited an increased volume of both subcutaneous and visceral fat. Accordingly, epididymal fat mass doubled in AKO mice compared to WT (Figure 2G), while there was no difference in lean body mass (Figure S1). Given that PPARγ plays a central role in the promotion of adipogenesis, these results suggest constitutive activation of adipose tissue PPARγ. Consistent with this interpretation, Figure 2H shows increased expression of the adipogenic PPARγ response genes FAS, ACC, SREBP1c, SCD1 and SCD2 in AKO adipose tissue (Djaouti et al., 2010; Lessard et al., 2007; Paton and Ntambi, 2009; Sugii et al., 2009).

Figure 2. Obese phenotype of adipocyte specific NCoR KO (AKO) mice.

Figure 2

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(A) Photograph, (B) Body weight, (C) Food intake, (D) Coronal section views of 3D MRI scan, (E) Subcutaneous fat mass, (F) Visceral fat mass, (G) Epi-WAT weight, and (H) Adipogenic gene expression levels in Epi-WAT. Values are expressed as mean ± SEM, n=8-10 in B, C, E- H, * P<0.05, ** P<0.01 for AKO versus WT. See also Table S2 and Figure S1.

Deletion of NCoR in adipose tissue protects against HFD-induced systemic insulin resistance

Obesity leads to glucose intolerance as well as insulin-resistance in adipose tissue, liver, and muscle. Therefore, we assessed glucose homeostasis and insulin sensitivity in lean chow-fed and HFD-fed WT and AKO mice. No changes in glucose or insulin tolerance, BW, fat pad weight or fasting insulin level, were noted in the lean, chow-fed mice between genotypes (Figure S1). In contrast, in the context of HFD/obesity, marked phenotypic changes in glucose and insulin homeostasis emerged in the AKO mice. Upon glucose and insulin tolerance testing, the AKO mice showed an enhanced hypoglycemic response to the injected insulin (Figure 3C), as well as improved glucose tolerance compared to controls (Figure 3D). In aggregate, these results argue strongly for improved systemic insulin sensitivity as a result of adipocyte NCoR deletion.

Figure 3. Improved glucose metabolism and insulin sensitivity in AKO mice.

Figure 3

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(A) Fasting blood glucose levels (B) Fasting blood insulin levels. (C) Insulin tolerance tests. (D) Glucose tolerance tests. (E) Glucose infusion rate (GIR) during hyperinsulinmic euglycemic clamp. (F) Glucose disposal rate (GDR). (G) insulin-stimulated glucose disposal rate (IS-GDR). (H) Basal hepatic glucose production (HGP). (I) Percent suppression of HGP by insulin (HGP suppression). (J) Percent suppression of free fatty acid levels (FFA suppression). (K) Insulin-stimulated phospho- Akt (Ser473) in liver, (L) muscle, and (M) adipose tissue. Values are expressed as mean ± SEM, n=8-10 in A- D, n=6 in E-J, * P<0.05, ** P<0.01 for AKO versus WT. See also Figure S1 and S2.

To more accurately quantify in vivo insulin action and to assess tissue specific sites of insulin sensitivity, hyperinsulinemic/euglycemic clamp studies were performed. The amount of exogenous glucose required to maintain euglycemia (GIR) and the glucose disposal rate (GDR) during the clamp studies was substantially higher in the AKO mice compared to WT (Figures 3E&F). The insulin stimulated glucose disposal rate (IS-GDR), which primarily reflects skeletal muscle insulin sensitivity and insulin's ability to suppress hepatic glucose production (HGP), which reflects hepatic insulin sensitivity, was also improved in the AKO group (Figures 3G-I). Finally, the ability of insulin to suppress plasma FFA levels provides an indication of adipose tissue insulin sensitivity, and FFA suppression was nearly twice as great in the AKO mice compared to WT (Figure 3J). We also harvested fat, liver and muscle tissue for ex vivo measurements of insulin action. As seen in Figures 3K-M, insulin stimulated Akt phosphorylation was augmented in all 3 tissues in the AKO mice compared to WT. Livers of the obese AKO mice were markedly less steatotic compared to WT by HE staining, with reduced expression of the lipogenic genes, SREBP1c, FAS, and ACC (Figure S2). Similarly, in muscle we found a marked decrease in TAGs, DAGs, and ceramides in the AKO group (Figure S2). Taken together, these data demonstrate that deletion of NCoR from adipocytes leads to a marked improvement in systemic insulin sensitivity, and these effects were expressed in all three major insulin target tissues, muscle, liver, and adipose tissue.

Adipocyte function in WT and AKO mice

Obesity represents an enlargement of adipose tissue to store excess energy and this is mostly accomplished by hypertrophy of fat cells, with hyperplasia generally assuming a smaller role. In chow-fed animals, the average epididymal adipocyte diameter was 60 μm, and in age-matched HFD/obese mice, fat cell diameter increased dramatically to 219 μm in WT mice. Interestingly, in the AKO group, the increase in adipocyte size was less on HFD (169 μM) (Figure 4A). This equates to a 54% decrease in average adipocyte volume in the AKO mice and per gm of adipose tissue there are 19.8×l04 fat cells in WT compared to 43×l04 cells in the AKO animals. This indicates that hyperplasia plays a greater role in adipose tissue expansion in AKO mice, where adipocyte PPARγ is constitutively activated, and this increase in fat cell number accounts for the greater adiposity in the AKO group. Interestingly, adipose tissue PPARγ levels (Figure 4B) were increased in the HFD AKO mice, providing an additional potential mechanism for enhanced PPARγ signaling. In the lean chow fed mice, no changes in PPARγ gene expression were noted in any of the adipose tissue depots examined (Figure S3). We measured tissue cathepsin D activity, perilipin content, and tunnel staining and found that levels of cathepsin D and perilipin were lower in the AKO mice, as was tunnel staining, indicative of lower rates of apoptosis/necrosis (Figures. 4C-E). Brown adipose tissue (BAT) mass and BAT related gene expression were unchanged in the AKO mice (Figure S4).

Figure 4. Increased adipogenesis and reduced adipocyte cell death in AKO mice.

Figure 4

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(A) Adipose tissue was stained for caveolin and adipocyte diameter was quantified. (B) Relative mRNA levels of PPARγ, (C) Cathepsin D activity, (D) Perilipin staining, (E) Tunnel staining, and (F) Circulating adipokine (leptin, high molecular weight-adiponectin, resistin and PAI-1) levels in control and AKO mice. (G) Blood free fatty acid (FFA) levels. (H) Basal and insulin stimulated 2-DOG glucose uptake in primary adipocytes. Values are expressed as mean ± SEM, n=8-10 in B, C, F and G. n=5 in H, * P<0.05, ** P<0.01 for AKO versus WT. See also Table S2 and Figure S3.

Besides storing lipids, adipose tissue functions as an endocrine organ, secreting a large number of polypeptide hormones, termed adipokines, which can modify systemic glucose homeostasis and insulin sensitivity. We measured adipokine levels in the AKO mice and found lower levels of leptin, resistin, PAI-1, but higher levels of HMW-adiponectin (Figure 4F), indicating that deletion of NCoR in adipocytes led to an adipokine profile favoring insulin sensitivity.

In adipose tissue, insulin promotes the uptake and storage of fatty acids in the form of triglycerides and inhibits lipolysis of stored triglycerides. Circulating levels of free fatty acids (FFA) were lower in the AKO mice (Figure 4G), suggesting lower rates of lipolysis in the adipose tissue. We also measured insulin stimulated glucose uptake in primary adipocytes and as seen in Figure 4H, the ability of insulin to enhance glucose transport was 30% higher in AKO versus WT adipocytes. This directly demonstrates cell autonomous insulin sensitivity in AKO cells, consistent with the insulin signaling data provided in Figure 3M. Conditioned media (CM) harvested from LPS treated RAW264.7 macrophages contains high levels of secreted cytokines which can inhibit insulin stimulated glucose transport. Interestingly, when adipocytes were treated with RAW 264.7 cell CM, the expected inhibition of glucose transport was observed in WT adipocytes, but this effect of CM was attenuated in the AKO cells (Figure S3).

Macrophage infiltration and inflammation in adipose tissue

Large numbers of adipose tissue macrophages (ATMs) accumulate in obesity and substantial evidence exists pointing to an etiologic role for these cells in the development of chronic tissue inflammation and insulin resistance/diabetes (Li et al., 2010; Lumeng et al., 2007; Nguyen et al., 2007; Weisberg et al., 2003; Xu et al., 2003). Thus, SVCs of epi-WAT depots from the two groups were isolated and stained with F4/80 and CD11b antibodies. This immunohistochemical analyses showed a large increase in F4/80 and CD11b double positive macrophages in the WT mice after 17 weeks of HFD while ATM accumulation was much less in the AKO group (Figure 5A). Consistent with this, adipose tissue F4/80 mRNA expression was lower in the AKO mice (Figure 5B). ATMs often surround dead adipocytes to form crown like structures (CLS) and there were fewer CLS in the AKO mice compared to WT mice (Figure 5C). These findings demonstrate that ATM content is diminished in the AKO animals, despite the fact that they are more obese.

Figure 5. Decreased adipose tissue inflammation in AKO mice.

Figure 5

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(A) FACS analysis of F4/80+/CD11b+ cells in SVF. (B) Relative mRNA levels of the macrophage marker F4/80 in Epi-WAT. (C) F4/80 immuno staining in epi-WAT. (D) FACS analysis of F4/80+/CD11b+/CD11c+ cells in SVF. (E) Relative mRNA levels of the proinflammatory M1- like macrophage marker CD11c in Epi-WAT. (F) Relative mRNA levels of inflammatory and anti-inflammatory cytokines in Epi-WAT. (G) Effect of conditioned medium (CM) from WT and AKO primary adipocytes on macrophage chemotaxis. (H) In vivo PKH26 flourescently labeled macrophage tracking in WT and AKO mice on HFD. (I) Subpopulations of recruited PKH26+ macrophages. Values are expressed as mean ± SEM, n=6 in A, B, D and E, n=8 in F, n=5-6 in G-I, * P<0.05, ** P<0.01 for AKO versus WT. See also Table S2 and Figure S5.

ATMs are heterogeneous, and there are at least two subpopulations of F4/80+, CD11b+ ATMs, one of which is positive and the other negative for CD11c. The CD11c+ (triply positive, F4/80+, CD11b+, CD11c+ cells), Ml-like, macrophages account for the majority of the increase in ATMs in obesity and overexpress pro-inflammatory cytokines compared with the CD11c-, M2-like, ATMs (Hevener et al., 2007; Li et al., 2010; Lumeng et al., 2007; Nguyen et al., 2007). As shown in Figures 5D&E, there was a marked decrease in CD11c+ ATMs in the AKO mice compared to WT. Most proinflammatory genes, including TNFα, IFNγ, IL-1β, iNOS, IL12p40, COX2, and MCP-1, were reduced in the AKO mice (Figure 5F), while expression of the non-inflammatory M2 marker genes, arginase and Mgl2 was increased. To expand this analysis, we measured these mRNAs in adipocytes compared to SVCs (Figure S5). These results were quite consistent with the whole adipose tissue data in that proinflammatory gene expression was generally decreased in both the SVCs and adipocytes from AKO mice.

To assess the mechanisms underlying the decreased ATM content in AKO mice, we measured the effects of CM from WT and AKO primary adipocytes to induced macrophage chemotaxis. AKO CM-mediated in vitro chemotaxis was markedly decreased compared to WT CM (Figure 5G), fully consistent with the reduced ATM content in AKO adipose tissue. We studied this concept further by directly measuring macrophage migration into adipose tissue using an in vivo macrophage tracking technique. With this approach, circulating monocytes were obtained from a WT donor mouse and labeled with fluorescent PKH26 dye ex vivo. The labeled monocytes were then injected into recipient HFD WT or HFD AKO mice. As seen in Figure 5H, there was a substantial decrease in labeled macrophage appearance in adipose tissue in the AKO mice. These data were even more revealing when we examined the subpopulations of labeled macrophages between the genotypes. With this analysis, (Figure 5I) there is an even greater decrease in the number of recruited macrophages which express CD11c, while, at the same time, there is an increase in the CD11c- macrophage population in the AKO mice.

AKO mice are relatively refractory to PPARγ stimulation

TZD treatment leads to increased insulin sensitivity, and we assessed this by treating HFD WT and HFD AKO mice with the PPARγ agonist Rosiglitazone for 3 weeks, followed by hyperinsulinemic-euglycemic clamp studies. The data in Figure 6 demonstrate that the WT mice displayed increased muscle (Figure 6A), liver (Figure 6B) and adipose tissue (Figure 6C) insulin sensitivity after Rosiglitazone treatment. The AKO mice were already insulin sensitive prior to drug treatment (Figure 3) and Rosiglitazone had no further effects in muscle and fat, but did induce even greater insulin sensitivity in liver (Figure 6). This suggests that the AKO mice are relatively resistant to exogenous PPARγ stimulation, most likely because adipocyte PPARγ target genes are already derepressed by the NCoR deletion. Unwanted side effects of TZD treatment include hemodilution and increased heart weight. Interestingly, neither were observed in the AKO mice, while, as expected, the effects of Rosiglitazone treatment to cause hemodilution were the same in both genotypes (Figure S4).

Figure 6. Effects of Rosiglitazone treatment and PPARγ serine273 phosphorylation.

Figure 6

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(A) IS-GDR in mice with or without Rosiglitazone treatment. (B) Percent suppression of HGP (HGP suppression). (C) Percent suppression of free fatty acid levels (FFA suppression). (D) Phospho- PPARγ (Ser273) and phospho-CDK5 (Tyr15) levels in epi-WAT. (E) Phospho-PPARγ levels in primary adipocytes. (F) Phospho-PPARγ and phospho-CDK5 levels in epi-WAT with or without Rosiglitazone treatment. (G) Phospho-Rb levels in epi-WAT. (H) TNFα–induced PPARγ ser 273 phosphorylation in primary adipocytes. (I) Mammalian two-hybrid assays in HEK293T cells using Gal4-PPARγ and VP16-NCoR-N (N-terminal 1-740 aa), VP16-NCoR-M (middle 742-1798 aa) or VP16-NCoR-C (C-terminal 1803-2439 aa). Also shown is the effect of Rosiglitazone on the PPARγ-NCoR-C interaction. (J) co-immunoprecipitation of CDK5 with PPARγ in epi-WAT. (K) Effects of NCoR, or the NCoR C-terminal domain, on TNFα -induced PPARγ ser 273 phosphorylation. (L) Mammalian two-hybrid assays using Gal4-CDK5 and VP16-PPARγ. (M) Effect of NCoR and Rosiglitazone on the PPARγ-CDK5 interaction. Values are expressed as mean ± SEM, n=6 in A- C, n=4-5 in D- H, and J * P<0.05, ** P<0.01 for AKO versus WT. See also Figure S4.

To further explore the mechanisms of this relative refractoriness to TZD treatment, we examined PPARγ expression and its phosphorylation state in adipose tissue. Choi et al. have recently shown that CDK5-mediated phosphorylation of PPARγ at serine 273 is an important regulatory event (Choi et al., 2010). In turn, PPARγ agonist treatment leads to dephosphorylation of PPARγ serine 273 and dephosphorylation at this site confers an active transcriptional program, consistent with an insulin sensitive state. In our current studies, we found that PPARγ mRNA (Figure 4B) and protein expression were increased in the AKO mice in adipose tissue extracts (Figure 6D) or in primary adipocytes (Figure 6E). Importantly, a substantial decrease in serine 273 phospho-PPARγ was also observed in both adipose tissue and primary adipocytes, and the ratio of phospho-PPARγ to total PPARγ protein was markedly reduced in the HFD-fed AKO mice compared to WT. It is unlikely that this decreased PPARγ phosphorylation was due to decreased CDK5 activity, since CDK5 protein levels and, more importantly, phospho-CDK5 levels, were comparable between WT and AKO mice (Figures 6D&F). Furthermore, phosphorylation of Rb, another cellular substrate of CDK5, was intact in the AKO cells (Figure 6G). Rosiglitazone treatment (Figure 6F) led to decreased PPARγ serine 273 phosphorylation in WT animals, as previously reported by Choi et al (Choi et al., 2010). Since non-phosphorylated PPARγ has insulin sensitizing actions, the finding that NCoR depletion inhibits this phosphorylation event is important and could contribute to the AKO phenotype. To further explore the mechanisms for this effect, we treated WT and AKO primary adipocytes with TNFα. TNFα is known to activate CDK5, and Figure 6H shows that TNFα treatment led to an increase in serine 273 PPARγ phosphorylation in WT adipocytes. Importantly, TNFα did not increase serine 273 PPARγ phosphorylation in AKO cells, demonstrating a cell autonomous effect of NCoR to facilitate CDK5-mediated PPARγ phosphorylation.

The molecular events underlying PPARγ phosphorylation were assessed with a series of co-immunoprecipitation and mammlian two-hybrid assays. In these studies, we used a luciferase reporter assay driven by Gal4-PPARγ coupled with VP16-NCoR constructs, encompassing NCoR residues 1-740 (NCoR-N), 742-1798 (NCoR-M), and 1803-2439 (NCoR-C). The data clearly show a direct interaction between PPARγ and NCoR, and the NCoR interaction site is located in the RID-containing C-terminus. Rosiglitazone treatment strongly inhibits this interaction almost to base line values (Figure 6I). We also performed co-immunoprecipitation experiments and confirmed earlier reports (Yu et al., 2005), that PPARγ co-precipitates with NCoR in WT cells (data not shown). More importantly, we also found that PPARγ readily precipitates with CDK5 in WT cells with ∼55% less PPARγ co-precipitated in the AKO cells (Figure 6J). Rosiglitazone causes dissociation of the PPARγ•NCoR complex (Figure 6I) and this leads to decreased CDK5/PPARγ co-precipitation in WT cells (Figure 6J). Transient transfection assays showed that full length NCoR and the NCoR C-terminus enhance TNFα stimulated CDK5 phosphorylation of PPARγ serine 273 in HEK293T cells (Figure 6K). In the two-hybrid system (Figure 6L) we used Gal4-CDK5 and VP16-PPARγ to show direct association between PPARγ and CDK5. Expression of NCoR increases this interaction, while Rosiglitazone treatment (which causes NCoR dismissal) decreases this interaction (Figure 6M). In the two-hybrid system, we also found no evidence for a direct interaction between NCoR and CDK5 (data not shown). Taken together, these results indicate that NCoR directly interacts with PPARγ and, through an allosteric effect, enhances the ability of PPARγ to associate with CDK5. When CDK5 activity is stimulated, as with TNFα treatment in vitro or obesity-induced adipose tissue inflammation in vivo, PPARγ serine 273 phosphorylation is induced.

Studies of Gene Expression

Since NCoR can interact with other transcription factors, in addition to PPARγ, we compared gene expression profiles in epi-WAT from HFD-fed WT and HFD-fed AKO mice. Fold change was calculated against the transcript levels of the WT group. The results revealed that 163 genes were up-regulated and 107 genes were down-regulated in the AKO group compared to WT. The most significantly enhanced pathway in the up-regulated group was the annotated PPARγ signaling pathway. qPCR was used to better quantitate a group of PPARγ response genes (Figure 7) in addition to those already shown in Figure 2H. These results demonstrate that these genes were uniformly up-regulated in the AKO mice compared to WT, and that Rosiglitazone treatment led to increased expression of these genes in WT animals, but had no effect to further enhance expression in the AKO mice. These results fortify the conclusion that adipocyte NCoR deletion leads to an activated PPARγ transcriptional state which is not further enhanced by PPARγ ligand (TZD) treatment. Consistent with this, RGS2 is a gene known to be downregulated by PPARγ activation and Figure 7B shows that RGS2 expression is decreased in the AKO mice and that Rosiglitazone treatment decreases RGS2 expression in WT but not AKO animals. Additional KEGG analysis revealed that up-regulated genes were also significantly enriched in functionally annotated pathways including: fatty acid metabolism, insulin signaling pathway, and mitochondria and lipid metabolic process. Down-regulated genes were significantly enriched for functional annotations such as natural killer cell-mediated toxicity and immune system process. The genes associated with the enriched functional pathways mentioned above are shown in Table S1.

Figure 7. mRNA levels of PPARγ target genes in epi-WAT.

Figure 7

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(A) mRNA levels of up-regulated genes in epi-WAT. (B) mRNA level of a down-regulated gene (RGS2). (C) SRC3 occupancy on PPARγ responsive elements (PPRE) in the Pepck/Gpd1 promoters. Values are expressed as mean ± SEM, n=6-8 in A and B, * P<0.05 for AKO versus WT. See also Table S1 and S2.

To probe the mechanisms of ligand-independent gene upregulation in AKO cells, we conducted ChIP assays for the PEPCK promoter. As seen in Figure 7C, Rosiglitazone treatment led to the expected increase in co-activator (SRC3) recruitment to the promoter in WT cells, whereas, in AKO cells, SRC3 occupancy was already high in the absence of ligand, with no further increase due to Rosiglitazone. This pattern corresponds quite closely to the gene expression results shown in Figure 7A (first box). In contrast, Gpd1 is a PPARγ-induced gene that was not constitutively upregulated in the AKO cells and ChIP assays showed the classical pattern of Rosiglitazone-mediated SRC3 recruitment in both WT and AKO cells.

Discussion

Adipose tissue serves as an integrator of various physiological pathways and is at the key cross roads for regulation of energy balance and glucose homeostasis (Cusi, 2010; Goossens, 2008; Halberg et al., 2008; Kahn and Flier, 2000; Qatanani and Lazar, 2007). Nuclear receptors are a family of transcription factors which modulate many aspects of biologic function, including glucose and energy homeostasis (Evans et al., 2004; Francis et al., 2003; Tontonoz and Spiegelman, 2008). NCoR is a major co-repressor of nuclear receptor function (Glass and Rosenfeld, 2000) and, therefore, should be a regulator of energy and glucose balance. In the current studies, we have generated adipocyte specific NCoR knockout mice and have conducted detailed in vivo and in vitro studies of adipocyte biology, obesity, inflammation, and insulin resistance. We find that deletion of NCoR from adipocytes leads to a phenotype which phenocopies constitutive PPARγ activation. The AKO animals become more obese on HFD than WT mice, due to increased adipocyte hyperplasia. Despite the increased adiposity, the AKO mice demonstrate decreased ATM content, reduced tissue inflammation, cell autonomous adipocyte insulin sensitivity and enhanced systemic insulin sensitivity. Interestingly, they are also relatively refractory to Rosiglitazone treatment. Although NCoR can serve as a co-repressor for multiple nuclear receptors, it is quite striking that the major phenotype in AKO mice can be traced to unrestrained PPARγ activity. This allows us to infer that in adipocytes, the dominant function of NCoR is to repress PPARγ. Furthermore, since genetic deletion of NCoR in adipocytes is the initiating event in our model, these studies demonstrate that unleashing PPARγ only in adipose tissue is sufficient to produce the systemic effects seen with in vivo TZD treatment, indicating the importance of adipose tissue for the metabolic effects of these drugs.

The AKO mice exhibited enhanced systemic insulin sensitivity as demonstrated by improved oral glucose tolerance with a reduction in hyperinsulinemia, and a greater hypoglycemic response to insulin during the insulin tolerance test. This increased insulin sensitivity was quantitated using the hyperinsulinemic/euglycemic clamp, as well as ex vivo tissue analyses. Importantly, the improved insulin sensitivity was not simply confined to adipose tissue, since increased hepatic and skeletal muscle insulin sensitivity was clearly demonstrated in the glucose clamp studies, with a 95% increase in IS-GDR (skeletal muscle insulin sensitivity) and 136% increase in hepatic insulin sensitivity (HGP suppression). These in vivo findings were strongly supported by the tissue measures of insulin signaling which showed improved insulin-stimulated Akt phosphorylation in all three insulin target tissues in the AKO animals.

Our studies also explored the physiologic mechanisms underlying the amelioration of insulin resistance due to adipocyte NCoR deletion. For example, it is well known that adipocytes secrete adipokines which can have systemic endocrine effects to modulate the tone of insulin action. We found that circulating levels of HMW adiponectin were increased, whereas, resistin levels were decreased in the AKO mice, and this combination could certainly improve insulin resistance. The AKO adipocytes also displayed increased insulin stimulated glucose transport compared to WT primary adipocytes. Furthermore, the effect of macrophage CM to cause insulin resistance in adipocytes was partially abrogated in the AKO cells similar to a TZD-treated state. This demonstrates increased cell autonomous adipocyte insulin sensitivity, providing an additional mechanism for improved in vivo insulin resistance.

It has been widely reported that chronic tissue inflammation can be a major cause of insulin resistance and that macrophage-mediated proinflammatory effects are an underlying mechanism (Donath and Shoelson, 2011; Hotamisligil and Erbay, 2008; Olefsky and Glass, 2010). Here we show that in HFD/obese mice, ATM accumulation was reduced in the AKO group and this was accompanied by decreased expression of proinflammatory genes in the adipose tissue. Interestingly, one of the important chemokines released by adipocytes is MCP-1, and PPARγ activation is known to inhibit adipocyte MCP-1 expression and secretion. We found decreased levels of MCP-1 in adipose tissue from the AKO mice, consistent with the activated PPARγ phenotype and this could provide an explanation for the decreased ATM numbers and reduced levels of inflammation. Indeed, we provide direct evidence consistent with this idea by showing that CM harvested from AKO compared to WT primary adipocytes has a much lower capacity to stimulate macrophage chemotaxis. In vivo macrophage tracking studies were also consistent with this concept. Here we found that when fluorescently labeled monocytes were injected into recipient HFD WT or AKO mice, a substantial decrease in macrophage appearance in adipose tissue was observed in the AKO animals. Taken together, these data suggest the overall idea that adipocyte NCoR deletion leads to decreased chemokine secretion from adipocytes, causing reduced ATM content and decreased inflammation, contributing to the enhanced state of insulin sensitivity.

It is of interest that the ultimate phenotype of the recruited ATMs is substantially different in the WT versus AKO mice. In WT mice, >90% of the fluorescently labeled recruited ATMs assume the Ml-like, CD11c+ proinflammatory phenotype, whereas, only ∼50% of the recruited ATMs in the AKO mice become CD11c+. This means that in the AKO mice there is a 6-fold increase in the proportion of these recruited ATMs which become M2-like, CD11c- ATMs. Thus, not only are the chemotactic signals reduced in AKO adipose tissue causing decreased total ATM migration, but differences in tissue cues also promote a higher degree of polarization to the M2-like ATM phenotype.

We were also able to study the WT and AKO mice after Rosiglitazone treatment and found that the predicted effects of this drug to improve insulin sensitivity were readily demonstrated in the HFD/obese WT mice. On the other hand, the HFD AKO mice were refractory to the effects of TZD treatment in adipose tissue and muscle. This is consistent with the PCR and gene array results which showed that the PPARγ transcriptional program is fully activated in the AKO adipose tissue and that Rosiglitazone treatment has very little additional effect. On the other hand, a clear improvement in hepatic insulin action was observed in the Rosiglitazone-treated AKO mice. Since the transcriptional effects of Rosiglitazone action involve dismissal of NCoR from PPARγ target genes, this mechanism can not be operative in adipocytes from the AKO mice. However, since NCoR expression was not altered in liver, the Rosiglitazone-mediated improvement in hepatic insulin sensitivity could still occur. With this in mind, it is interesting to note that, while Rosiglitazone treatment led to enhanced hepatic insulin action in the AKO mice, pretreatment hepatic insulin sensitivity was already greater in the AKO animals compared to WT. This indicates that in vivo TZD-induced hepatic insulin sensitization includes both direct drug effects in the liver and indirect effects, most likely originating in adipose tissue.

TZD treatment causes fluid retention as manifested by hemodilution, edema, and often times increased heart weight. As measured by hematocrit and heart weight, the AKO mice do not show signs of hemodilution (Figure S4C and D), demonstrating that the site of action of TZDs to cause edema is outside of the adipocyte, most likely in the kidney, where NCoR and the PPARγ system are fully preserved in our mice. Rosiglitazone causes the expected effect on hemodilution in both genotypes (Figure S4C), also consistent with a site of action outside of the adipose tissue. These results suggest that adipocyte specific stimulation of the PPARγ program could be an effective means to avoid some of the unwanted side effects associated with classical TZD treatment.

An important finding in these studies involves the status of PPARγ phosphorylation at serine 273. Choi et al. have demonstrated that CDK5 can phosphorylate PPARγ at position 273 (Choi et al., 2010), whereas, PPARγ agonists lead to decreased phosphorylation at this site. They have also shown that the dephosphorylated form of PPARγ is transcriptionally active, inducing a gene signature profile conducive to insulin sensitization. This finding prompted us to examine the status of PPARγ serine 273 phosphorylation in our studies. We found that serine 273 phosphorylation was decreased in whole adipose tissue and in isolated adipocytes from the AKO mice compared to WT. This difference was not due to reduced CDK5 activity, since the amount of phospho-CDK5 in adipose tissue and the phosphorylation state of another CDK5 substrate, Rb, was comparable between the two groups. From these findings, we infer that NCoR is necessary for CDK5-mediated PPARγ ser 273 phosphorylation. This may also help explain why PPARγ agonists cause decreased PPARγ serine 273 phosphorylation, since PPARγ agonists dismiss NCoR from the PPARγ transcriptional complex. This decrease in PPARγ serine 273 phosphorylation is likely an important factor in the insulin sensitive phenotype in these mice.

Given the potential importance of this finding to decreased insulin resistance, we conducted experiments to elucidate the mechanisms for the reduced PPARγ phosphorylation in the absence of NCoR. TNFα is an activator of CDK5, and Choi et al. have demonstrated that TNFα treatment of adipocytes increases serine 273 PPARγ phosphorylation. In the current studies, we found that TNFα treatment of WT primary adipocytes led to increased serine 273 PPARγ phosphorylation, whereas, TNFα had no such effect in AKO cells. This demonstrates that the ability of adipocyte NCoR deletion to decrease serine 273 PPARγ phosphorylation is a cell autonomous effect. Furthermore, we show that PPARγ co-precipitates with CDK5, demonstrating the presence of these two proteins in a molecular complex. Importantly, co-precipitation of PPARγ with CDK5 was markedly decreased in the AKO cells. Both two-hybrid and Co-IP studies show a direct interaction between NCoR and PPARγ, and this interaction is inhibited by Rosiglitazone treatment. We also show that NCoR enhances TNFα stimulated CDK5-mediated PPARγ serine 273 phosphorylation, while Rosiglitazone treatment, which dissociates NCoR from the complex, inhibits this phosphorylation event. In the two-hybrid system, the data show a direct interaction between PPARγ and CDK5, and NCoR has a substantial effect to enhance this association while Rosiglitazone treatment attenuates it. In contrast to the strong inteactions between PPARγ and NCoR, and PPARγ and CDK5, we found only a negligible direct interaction between NCoR and CDK5. We also observed that TNFα treatment does not increase CDK5/PPARγ association, consistent with the view that the TNFα effect is conferred by augmenting the enzymatic activity of CDK5, rather than its association with its PPARγ target protein. In sum, when NCoR is decreased by genetic deletion or Rosiglitazone treatment, the CDK5/PPARγ interaction is attenuated and subsequent phosphorylation of PPARγ serine 273 is reduced. When NCoR expression is enhanced, the interaction between CDK5 and PPARγ is augmented, as is PPARγ serine 273 phosphorylation. All of these results are consistent with the concept that the NCoR interaction with PPARγ produces an allosteric effect to enhance the ability of PPARγ to associate with CDK5.

With respect to the constitutively upregulated genes, a transcriptional mechanism would predict low basal co-activator occupancy of the promoter in WT cells with an increase upon Rosiglitazone treatment. Due to NCoR deletion in AKO cells, co-activator occupancy should be high in the absence of ligand with no further increase due to Rosiglitazone. This is precisely the pattern observed in our ChIP studies for SRC3 on the PEPCK promoter (Figure 7C). In contrast, Gpd1 is a PPARγ gene which was not upregulated in the AKO cells and ChIP assays showed the classical pattern of Rosiglitazone-induced co-activator recruitment in both WT and AKO cells. The degree of insulin sensitivity in adipose tissue and muscle was the same in untreated AKO mice as in Rosiglitazone treated WT animals, and, therefore, it seems logical to suggest that the set of constitutively upregulated genes in the AKO mice, contains the key genes essential for TZD-mediated improvement of insulin sensitivity.

In these studies, we have shown that adipocyte-specific deletion of the co-repressor NCoR, specifically activates the PPARγ transcriptional program and promotes the non-phosphorylated PPARγ serine 273 state in these cells. This leads to increased adipogenesis, reduced adipose tissue ATM content and inflammation, improved cell autonomous adipocyte insulin sensitivity, changes in adipokine secretion, and increased systemic insulin sensitivity, all of which phenocopy the TZD treated in vivo state. Thus, while NCoR can co-repress multiple nuclear receptors, these data demonstrate selective cell type effects of this co-repressor, indicating that it's dominant action in adipocytes is to co-repress PPARγ. These results raise the possibility that NCoR may prove to be a useful target for future therapeutics in the treatment of Type 2 diabetes and other insulin resistant diseases.

Methods

Creation of control and adipocyte specific NCoR KO mice

Mice carrying floxed alleles of NCoR were provided by Dr. Johan Auwerx. These mice were backcrossed to the C57BL/6J strain for nine generations. Mice were bred with transgenic mice harboring Cre recombinase driven by aP2 promoter (He et al., 2003) to create the following genotypes: NCoRfl/fl (control), NCoRfl/fl-ap2Cre (AKO).

ITTs, GTTs, and hyperinsulinemic euglycemic clamp study

Glucose and insulin tolerance tests were performed on 6 hr fasted mice. For GTTs, animals were IP injected with dextrose (1 g/kg, Hospira, Inc), whereas for ITTs 0.5 units/kg of insulin (Novolin R, Novo-Nordisk) was IP injected. Blood was drawn at 0, 15, 30, 60, and 120 minutes after dextrose or 0, 15, 30, 60 and 90 minutes after insulin injection. Mouse clamps were performed as previously described (He et al., 2003; Li et al., 2010; Lu et al., 2010). Briefly, dual catheters (MRE-025, Braintree Scientific) were implanted in the right jugular vein and tunneled subcutaneously and exteriorized at the back of the neck. The mice were allowed to recover for 3 to 4 days before the clamp procedure. After 6 h fasting, the clamp experiments began with a constant infusion (5 μCi/hr) of D-[3-3H] glucose (Du Pont-NEN, Boston, MA). After 90 min of tracer equilibration and basal sampling at t = −10 and 0 min, glucose (50% dextrose, variable infusion; Abbott) and tracer (5 μCi/hr) plus insulin (6 mU/kg/min) were infused into the jugular vein. Blood from the tail vein was drawn at 10-min intervals and analyzed for glucose to maintain the integrity of the glucose clamp. Blood was taken at t = −10, 0 (basal), 110, and 120 (end of experiment) min to determine glucose-specific activity, insulin, and free fatty acids (FFA). Steady-state conditions (120 mg/dl ± 5 mg/dl) were confirmed at the end of the clamp by ensuring that glucose infusion and plasma glucose levels were maintained constant for a minimum of 30 min. HGP and GDR were calculated in the basal state and during the steady-state portion of the clamp. Tracer-determined rates were quantified by using the Steele equation (Steele, 1959). At steady state, the rate of glucose disappearance, or total GDR, is equal to the sum of the rate of endogenous glucose productions (HGP) plus the exogenous (cold) GIR. The IS-GDR is equal to the total GDR minus the basal glucose turnover rate.

Other methods

See Supplementary material.

Statistical analyses

Data are presented as the mean ± SEM For experiments involving two factors, data were analyzed by two-way ANOVA followed by Bonferroni post tests. Individual pair-wise comparisons were performed using student t test. The p value<0.05 was considered significant.

Supplementary Material

01
02

Highlights.

  1. Adipocyte specific NCoR KO leads to de-repression of PPARγ.

  2. CDK5-mediated phosphorylation of PPARγ serine 273 is decreased by NCoR deletion.

  3. NCoR is an adaptor protein for CDK5-PPARγ association and ser 273 phosphorylation.

  4. PPARγ de-repression leads to insulin sensitivity and decreased inflammation.

Acknowledgments

We thank Elizabeth J. Hansen for editorial assistance. We thank the Flow Cytometry Resource (Dennis Young) for FACS analysis at the Rebecca & John Moores Cancer Center and thank the UCSD Histology Core lab for technical help with processing tissue specimens and microscope analysis. This study was funded in part by the National Institutes of Health grants NIDDK DK033651 (J.M.O.), DK063491 (J.M.O.), DK 074868 (J.M.O.), DK059820 (J.A.) and EU Ideas program (ERC-2008-AdG-23118), the Swiss National Science Foundation, and the Eunice Kennedy Shriver NICHD/NIH through a cooperative agreement U54 HD 012303-25 as part of the specialized Cooperative Centers Program in Reproduction and Infertility Research.

Footnotes

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Supplementary Materials

01
NIHMS333422-supplement-01.pdf (347.8KB, pdf)
02
NIHMS333422-supplement-02.pdf (1.9MB, pdf)

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